Gram negative bacterial infection is a widespread problem in critically ill patients. The high mortality of sepsis is in part mediated by bacterial endotoxin (LPS), which activates mitogen-activated protein (MAP) kinases (e.g., p38, ERK 1/2, and JNK), and stimulates the release of proinflammatory cytokines (e.g., TNF and IL-1beta), nitric oxide, platelet-activating factor, and other products. Macrophage-derived cytokines have been implicated in mediating lethal endotoxemia, because inhibition of their production or activity attenuates the development of tissue injury in animal models. If delivered early enough, anti-TNF can be an effective therapy in experimental models of endotoxemia, but early treatment is difficult to achieve in the clinic. An alternative strategy would be to identify "late" macrophage mediators that may be clinically more accessible. We recently identified a ubiquitous protein, HMGB1 (formerly known as HMG-1), as a late mediator of endotoxin lethality (Science 1999, 285: 248-251). HMGB1 is released late by LPS-stimulated macrophages, and its serum levels increase significantly between 16 to 32 hours after exposure to endotoxin. Anti-HMGB1 antibodies significantly protect against lethal endotoxemia and LPS-induced acute lung injury, even when antibody administration is delayed until after the early TNF response. Purified recombinant HMGB1 induced the release of multiple cytokines (e.g., TNF, IL-1beta and IL-6) in macrophage/monocyte cultures, and promoted tissue injury and even lethality when administered into mice. However, the mechanisms underlying the regulation of HMG-1 release and action are still unknown. The first aim of the studies outlined in this proposal is to determine the roles of early pro-inflammatory cytokines (e.g., TNF, IL-1beta) and MAP kinase (e.g., p38 and ERK1/2) signaling pathways in regulation of LPS-induced HMG-1 release in macrophage/monocyte cultures. This will be accomplished by examining the effect of TNF- or IL-1beta-specific neutralizing antibodies, as well as MAP kinase-specific inhibitors or anti- sense oligonucleotides on LPS-induced HMGB1 release. The second aim of this proposal is to examine the role of HMGB1 receptor (e.g., RAGE) and MAP kinases (e.g., p38, ERK1/2, and JNK) in regulation of HMGB1-induced cytokine production in macrophage/monocyte cultures. We will examine whether HMGB1 will activate MAP kinases, and whether RAGE-specific neutralizing antibodies or anti-sense oligonucleotides will prevent HMGB1- induced TNF release. Answers to these questions will shed light on the mechanisms underlying regulation of HMGB1 release and action, and improve our understanding of mechanisms underlying regulation of the innate immune response in endotoxemia.